Porcine Bile Research Articles

Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile

Bifidobacterium breve 46, Bifidobacterium lactis 8:8 and Bifidobacterium longum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacterium pseudocatenulatum JCM 1200 were examined for acid and bile tolerance, prebiotic utilization and antimicrobial activity against four Clostridium difficile (CD) strains including the hypervirulent strain, PCR ribotype NAP1/027. B. lactis 8:8 and B. lactis JCM 10602 exhibited a high tolerance in MRSC broth with pH 2.5 for 30 min. B. breve 46 and B. lactis 8:8 remained 100% viable in MRSC broth with 5% porcine bile after 4 h. All six strains showed a high prebiotic degrading ability (prebiotic score) with galactooligosaccharides (GOS), isomaltooligosaccharides (IMOS) and lactulose as carbon sources and moderate degradation of fructooligosaccharides (FOS). Xylooligosaccharides (XOS) was metabolized to a greater extent by B. lactis 8:8, B. lactis JCM 10602, B. pseudocatenulatum JCM 1200 and B. longum 6:18 (prebiotic score >50%). All strains exhibited extracellular antimicrobial activity (AMA) against four CD strains including the CD NAP1/027. AMA of B. breve 46, B. lactis 8:8 and B. lactis JCM 10602 strains was mainly ascribed to a combined action of organic acids and heat stable, protease sensitive antimicrobial peptides when cells were grown in MRSC broth with glucose and by acids when grown with five different prebiotic-non-digestible oligosaccharides (NDOs). None of C. difficile strains degraded five prebiotic-NDOs. Whole cells of B. breve 46 and B. lactis 8:8 and their supernatants inhibited the growth and toxin production of the CD NAP1/027 strain.

Porcine feasibility and safety study of a new paclitaxel-eluting biliary stent with a Pluronic-containing membrane

Metal stents for malignant biliary obstruction are susceptible to occlusion by tumor ingrowth or overgrowth. Therefore, we previously reported our use of a metal stent covered with a paclitaxel-incorporated membrane giving an antitumor effect to prevent occlusion from tumor ingrowth. We have also developed a new generation of paclitaxel-eluting biliary stent using a membrane containing Pluronic F-127 for effective drug delivery. The aim of this study was to investigate the safety and efficacy of drug delivery for this newly developed stent in the biliary tract. Metal stents were coated with paclitaxel and various concentrations of Pluronic F-127 in phosphate-buffered saline solution. Stents containing varying concentrations were placed in the bile ducts of eight pigs divided as follows: group I, 0% Pluronic + 0% paclitaxel; group II, 0% Pluronic + 10% paclitaxel; group III, 10% Pluronic + 10% paclitaxel; group IV, 20% Pluronic + 10% paclitaxel. The histology of the porcine bile duct and the amount of paclitaxel in the porcine serum were examined. The amount of paclitaxel released was also measured in vitro. Histologic changes in the porcine biliary epithelium were acceptable in terms of safety, based on inflammatory cell infiltration and fibrotic reaction. No significant differences in histology were observed between the groups. In the porcine serum analysis, released paclitaxel was detected for 28 days with the 10% Pluronic concentration (group III). However, released paclitaxel was observed for only 7 days in groups II and IV. In the in vitro experiments, long-lasting release of paclitaxel was also noted from the stent with 10% Pluronic. The new paclitaxel-eluting stent with 10% Pluronic F-127 is safe and provides enhanced local drug delivery.

Bile stimulates cell surface hydrophobicity, Congo red binding and biofilm formation of Lactobacillus strains

Seventeen Lactobacillus strains were tested for cell surface hydrophobicity (CSH) using the salt aggregation test (SAT) and Congo red binding (CRB) assay. CRB was dependent on pH and ionic strength and was protease-sensitive. In the presence of 100 μg mL(-1) cholesterol, the CRB was significantly reduced. Autoaggregating (AA) Lactobacillus crispatus strains showed 50% more CRB than the reference strain, the curli-producing Escherichia coli MC4 100. CRB of L. crispatus 12005, L. paracasei F8, L. plantarum F44 and L. paracasei F19 were enhanced when grown in Man Rogosa Sharpe (MRS) broth with 0.5% taurocholic acid (TA) or 5% porcine bile (PB) (P < 0.05). CSH was also enhanced for the non-AA strains L. plantarum F44, L. paracasei F19 and L. rhamnosus GG when grown in MRS broth with 0.5% TA, 5% PB or 0.25% mucin, with enhanced biofilm formation in MRS broth with bile (P < 0.05). Two AA strains, L. crispatus 12005 and L. paracasei F8, developed biofilm independent of bile or mucin. In summary, under bile-stressed growth conditions, early (24-h cultures) biofilm formation is associated with an increase in hydrophobic cell surface proteins and high CRB. Late mature (72-h culture) biofilm contained more carbohydrates, as shown by crystal violet staining.

Abstract 1331: The BK5.erbB2 mouse as a novel model of esophageal adenocarcinoma

Abstract The incidence of esophageal adenocarcinoma (EAC) is rising rapidly throughout the Western world, and is associated with gastroesophageal reflux disease (GERD) and Barrett's esophagus (BE). BE is intestinal metaplasia of squamous esophageal epithelium, important clinically due to the fact that it increases an individual's chance of progression to EAC 30 to 125-fold higher than people without BE. Despite its clinical importance, the molecular pathogenesis of BE is poorly understood. One limitation in the study of BE pathogenesis has been the lack of suitable experimental models. There is a critical need to develop good preclinical models of BE. ErbB2 was reported to be amplified and overexpressed in BE, EAC, and esophageal squamous cell carcinoma (ESCC). However, the role of erbB2 in the development and progression of esophageal cancer is still unknown. In this study, we utilized BK5.erbB2 mice, in which erbB2 is overexpressed in epithelial cells under the control of the bovine keratin 5 promoter to determine whether the interaction between erbB2 and bile acids is important in the etiology of esophageal cancers. BK5.erbB2 mice (2 months of age, n=8) and age matched wild type mice (n=10) were treated with porcine bile extract (10mg/mouse in 200μL water), taurochenodeoxycholate (TCDC) (0.1μmol/mouse in 200μL water), or water control, 3 times weekly for 14 weeks by gavage. In the BK5.erbB2 mice, exophytic tumors in the forestomach, which has the same histological structure of esophagus, were observed in 20%, 33%, and 0% of mice treated with bile extract, TCDC, and water control, respectively. No tumors were observed in wild type mice. All tumors observed in BK5.erbB2 mice were diagnosed as SCC. Western blotting analysis demonstrated that the bile acid treatment increased the levels of p-EGFR, p-MAPK, and p-Akt in the forestomachs of the BK5.erbB2 mice. Furthermore, CDX2 (a marker of BE) was significantly expressed in BK5.erbB2 mice treated with bile extract or TCDC. No development of intestinal metaplasia in forestomach was found. We further investigated using a surgically manipulated reflux model. Eleven 7 week-old BK5.erbB2 and 5 wild type mice were given an esophagogastroduodenostomy. Mice were sacrificed at post-operative week 8, and examined histologically. Hyperkeratosis and intestinal metaplasia were observed at the anastomosis sites in both BK5.erbB2 and wild type mice. These histological changes were more prominent in BK5.erbB2 mice. Interestingly, dysplastic lesions were observed only in BK5.erbB2 mice. These results indicate that more persistent exposure of bile via surgery induce the development of not only SCC, but also precancerous lesions of adenocarcinoma in the esophagus/forestomach of BK5.erbB2 mice. The interaction between erbB2 and bile acids is important in esophageal tumor formation and intestinal metaplasia. A surgical model using BK5.erbB2 mice could possibly be a promising model for the study of the development of EAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1331. doi:1538-7445.AM2012-1331

Kinetic analysis of bile salt passage across a dialysis membrane in the presence of cereal soluble dietary fibre polymers

The kinetics of passage of a model bile salt and complete porcine bile across a dialysis membrane, in the presence and absence of two cereal-derived soluble dietary fibre polysaccharides, were studied as a model for passage across the unstirred water layer that lines the small intestine. A first-order kinetic analysis allowed rate coefficients to be derived which quantified the effectiveness of barley mixed linkage β-glucan and wheat arabinoxylan in retarding the transport of bile. For both, a model bile salt and complete porcine bile, rate coefficients decreased with both concentration and viscosity. A combination of viscosity and molecular interaction effects is suggested to control the effect of the two polysaccharides on the transport of bile.

Tu1664 Cholangioscopy With Image-Guided Intervention in the Human Bile Duct Using Scanning Fiber Endoscope System (Sfe): Results of First Clinical Experience

Currently available endoscopic imaging devices used in the biliary tree are low resolution with poor image quality, leading to inadequate evaluation of biliary tract disorders and limiting image guided therapy. However, a new ultrathin and flexible cholangioscope system has been developed and successfully demonstrated in a porcine bile duct. This mini-cholangioscope system uses a scanning fiber endoscope (SFE) as a forward-imaging guidewire, dimensions of 1.2-mm diameter and 3-m length. Full color video (500-line resolution at 30Hz) is the standard SFE imaging mode using spiral scanning of red, green, and blue laser light at low power.

Safety evaluation of self‐expanding metallic biliary stents eluting gemcitabine in a porcine model

Palliative biliary decompression by metal stent is the treatment of choice for unresectable malignant biliary obstruction; however, conventional stents provide only mechanical palliation and exert no anti-tumor effects. Gemcitabine (GEM) has been reported to be more effective in unresectable pancreatic cancer and biliary cancer compared with other chemotherapeutic drugs. We evaluated the safety of a GEM-eluting stent by analyzing histologic responses of the porcine bile duct. Stents containing GEM (0%, 10%, 15%, and 20% [w/v]) were surgically inserted into bile ducts of pigs (each group, n = 2). The animals were euthanized after 4 weeks, and the stented bile duct segment underwent gross and microscopic examination. Laboratory assay was performed for aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin, and gamma-glutamyl transferase (γ-GTP). Moderate to severe inflammation was observed in the bile ducts in contact with stents containing 15 and 20% GEM, compared with no inflammation with 0% GEM and mild inflammation with 10% GEM. Fibrous reactions observed in the submucosal layer did not differ among groups. Transmural necrosis and perforations were not observed in any animal. No abnormal laboratory test findings were directly caused by GEM. Our newly developed GEM eluting stents can be used safely in normal bile ducts. Our results indicated that 10% GEM produced mild histologic changes in the stented segment and adjacent tissue; this concentration may be appropriate for clinical application.

Effects of human and porcine bile on the proteome of Helicobacter hepaticus

BackgroundHelicobacter hepaticus colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, H. hepaticus must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of H. hepaticus modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry.ResultsThe 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of H. hepaticus were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of H. hepaticus in response to human or porcine bile.ConclusionsOverall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.

Influence of Preservation Solutions and Bile on a Human Bile Duct Epithelium Cell Line: An &lt;i&gt;In-Vitro&lt;/i&gt; Study

Introduction: Bile duct complications are common after liver transplantation. The impact of preservation solution is unclear. Aim: We investigated the impact of different preservation solutions with and without diluted bile on a human biliary tract carcinoma cell line. Methods: The human biliary tree carcinoma cell line SK-ChA-1 was cultured with either medium or University of Wisconsin solution (UW), histidine-tryptophane-ketoglutarate (HTK) solution, Celsior or physiological saline for 6h, 10h or 12h at 6℃- 8℃ and metabolic activity was measured by a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-test immediately, after 12h or 24h. Cells were also incubated in combination with diluted porcine bile. Results: Immediately after cold storage of 6h HTK and UW decreased metabolic activity whereas Celsior increased metabolic activity after 10h or 12h of cold ischemia. After 12h or 24h no major differences were found any more. Incubation with bile in combination with HTK, Celsior or NaCl decreased metabolic activity, whereas UW abolished this effect. Conclusion: On a cellular level differences between preservation solutions were found, especially in combination with bile. Further studies are warranted to determine whether this results in clinically significant differences in biliary complications.

Transport Across Caco-2 Cell Monolayer and Sensitivity to Hydrolysis of Two Anxiolytic Peptides from αs1-Casein, α-Casozepine, and αs1-Casein-(f91–97): Effect of Bile Salts

α-Casozepine and f91-97, peptides from α(s1)-casein, display anxiolytic activity in rats and may have to cross the intestinal epithelium to exert this central effect. We evaluated their resistance to hydrolysis by the peptidases of Caco-2 cells and their ability to cross the cell monolayer. To mimic physiological conditions, two preparations of bile salts were used in noncytotoxic concentrations: porcine bile extract and an equimolar mixture of taurocholate, cholate, and deoxycholate. The presence and composition of bile salts appeared to modulate the peptidase activities of the Caco-2 cells involved (i) in the hydrolysis of α-casozepine, leading to much higher formation of fragments f91-99, f91-98, and f91-97, and (ii) in the hydrolysis of f91-97, leading to lower degradation of this peptide. Transport of α-casozepine across Caco-2 monolayer increased significantly, in the presence of bile extract, and of fragment f91-97, in the presence of bile salts.

Impact of bile salt adaptation of Lactobacillus delbrueckii subsp. lactis 200 on its interaction capacity with the gut

In a previous work, bile-salt-resistant derivatives were obtained from non-intestinal lactobacilli. The aim of this work was to investigate the impact of bile adaptation of Lactobacillus delbrueckii subsp. lactis 200 on morphology, surface properties, in vivo interaction capacity with the gut and ability to activate the gut immune response. Electron microscopy studies, growth kinetics in the presence of bovine and porcine bile, the capacity to deconjugate bile acids, hydrophobicity, autoaggregation and co-aggregation capacities were studied for the parental strain and its bile-resistant derivative in vitro. Additionally, survival in intestinal fluid, the interaction with the gut and the immunomodulating capacities were studied in mice. Bile salt adaptation conferred upon the adapted strain a higher capacity to withstand physiological concentrations of bile salts and greater survival capacity in intestinal fluid. However, bile salt exposure reduced cell hydrophobicity, autoaggregation and adhesion capacities, resulting in reduced persistence in the intestinal lumen and delayed capacity to activate the gut immune response. Insight into the effects of bile salts upon the interaction and immunomodulating capacity of lactobacilli with the gut is provided, relating in vitro and in vivo results.

In vitro lipolysis models as a tool for the characterization of oral lipid and surfactant based drug delivery systems

With the increasing interest in lipid and surfactant based drug delivery systems (LSBDDS) for oral delivery of poorly soluble drugs, the need for efficient development tools is emerging. In vitro lipolysis models, simulating the digestion in the small intestine, is a promising tool in this regard. Several different in vitro lipolysis models have been used for characterization of LSBDDS, all using porcine pancreatin as lipase source, and primarily differing in the addition scheme of calcium and the kind of bile acids employed. Both calcium and bile influence the lipolysis. Calcium have been used both as fixed addition at the beginning of the experiment and with a continuous addition during lipolysis. Both pure bile acids and crude porcine bile extract have been used. Lipolysis of LSBDDS will generate mixed micelles, as well as lamellar and hexagonal phases. These have been characterized by dynamic light scattering, cryogenic transmission electron microscopy and small angle X-ray scattering. The faith of drug during in vitro digestion of a LSBDDS is often studied by ultracentrifugation and quantification of drug in the different phases formed. Further, drug precipitated during in vitro lipolysis has been characterized by X-ray powder diffraction and polarized light microscopy.

Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

A serum-free, feeder cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1-wk-old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder cell layers of mitotically blocked mouse fibroblasts. In serum-free medium containing 1% DMSO and 1 μM dexamethasone, confluent monolayers of hepatocytes formed and could be maintained for several wk. Light and electron microscopic analysis showed hepatocytes with in vivo-like morphology, and many hepatocytes were sandwiched between the feeder cells. When isolated liver cells were cultured in medium without dexamethasone but with 0.5% DMSO, monolayers of cholangioctyes formed that subsequently self-organized into networks of multicellular ductal structures, and whose cells had monocilia projecting into the lumen of the duct. Gamma-glutamyl transpeptidase (GGT) was expressed by the cholangiocytes at their apical membranes, i.e., at the inner surface of the ducts. Cellular GGT activity increased concomitantly with the development of ductal structures. Cytochrome P-450 was determined in microsomes following addition of metyrapone to the cultures. In vivo-like levels of P-450s were found in hepatocyte monolayers while levels of P-450 were markedly reduced in cholangiocyte monolayers. Serum protein secretion in conditioned media was analyzed by Western blot and indicated that albumin, transferrin, and haptoglobin levels were maintained in hepatocytes while albumin and haptoglobin declined over time in cholangiocytes. Quantitative RT-PCR analysis showed that serum protein mRNA levels were significantly elevated in the hepatocytes monolayers in comparison to the bile ductule-containing monolayers. Further, mRNAs specific to cholangiocyte differentiation and function were significantly elevated in bile ductule monolayers in comparison to hepatocyte monolayers. The results demonstrate an in vitro model for the study of either porcine hepatocytes or cholangiocytes with in vivo-like morphology and function.

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures. The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.

Functional and phenotypic characterization of a protein from Lactobacillus acidophilus involved in cell morphology, stress tolerance and adherence to intestinal cells

Structural components of the cell surface have an impact on some of the beneficial attributes of probiotic bacteria. In silico analysis of the L. acidophilus NCFM genome sequence revealed the presence of a putative cell surface protein that was predicted to be a myosin cross-reactive antigen (MCRA). As MCRAs are conserved among many probiotic bacteria, we used the upp-based counterselective gene replacement system, designed recently for use in L. acidophilus, to determine the functional role of this gene (LBA649) in L. acidophilus NCFM. Phenotypic assays were undertaken with the parent strain (NCK1909) and deletion mutant (NCK2015) to assign a function for this gene. The growth of NCK2015 (ΔLBA649) was reduced in the presence of lactate, acetate, porcine bile and salt. Adhesion of NCK2015 to Caco-2 cells was substantially reduced for both stationary-phase (∼45 % reduction) and exponential-phase cells (∼50 % reduction). Analysis of NCK2015 by scanning electron microscopy revealed a longer cell morphology after growth in MRS broth compared to NCK1909. These results indicate a role for LBA649 in stress tolerance, cell wall division and adherence to Caco-2 cells.

Putative probiotic Lactobacillus spp. from porcine gastrointestinal tract inhibit transmissible gastroenteritis coronavirus and enteric bacterial pathogens.

A total of 310 bacterial strains isolated from the porcine gastrointestinal tract were tested for their activity against transmissible gastroenteritis (TGE) coronavirus and other enteric pathogens. Based on activity, the strains Probio-38 and Probio-37 were selected as potential probiotics and identified as Lactobacillus plantarum Probio-38 and Lactobacillus salivarius Probio-37 respectively by 16S rRNA gene sequencing. Supernatants of these strains inhibited TGE coronavirus in vitro in ST cells, without any cytopathic effect even after 72 h of incubation. Both the strains exhibited high survival in synthetic gastric juice. The strains were resistant to 5% porcine bile and exhibited antimicrobial activity against all the 13 enteric bacterial pathogens tested. These strains also exhibited resistance to most of the antibiotics analyzed. The inhibition of transmissible gastroenteritis coronavirus and enteric bacterial pathogens as well as the bile tolerance, high survival in gastric juice, and the antibiotic resistance indicate that the two isolated bacterial strains are ideal probiotic candidates for animal application after proper in vivo experiments.

Abstract 4176: Bile acid-promoted esophageal and forestomach carcinomas in BK5.erbB2 mice

Abstract Esophageal cancer is considered to be one of the most lethal cancers, with a 5-year survival rate of approximately 23% to 50% for patients, even after complete resection with lymph node dissection. There are two common forms of esophageal cancer: squamous cell carcinomas (ESCCs) and adenocarcinomas (EACs). It has been shown that the initial step in the development of EACs is the transformation of normal stratified squamous epithelium in the lower esophagus to metaplastic specialized columnar epithelium (SCE), a pathological condition caused by chronic gastroesophageal reflux of gastric acid and bile known as Barrett's esophagus (BE), and it is possible that this condition also leads to the development of ESCCs as well. Although it has been reported that erbB2 is strongly expressed in SCE, EACs, and ESCCs, the role of erbB2 in the development and progression of esophageal cancer is still unknown. Furthermore, the contribution of bile acids in the etiology of esophageal cancer has not been explored. In this study, to determine whether interaction between erbB2 and bile acids is important in the etiology of esophageal cancers, we utilized BK5.erbB2 mice, in which erbB2 is overexpressed in epithelial cells under the control of the bovine keratin 5 promoter. Eight two-month-old homozygous BK5.erbB2 mice and 8 wild type mice were initiated with N-nitrosomethylbenzylamine (NMBA, 50μg/mouse in 200μL water, twice weekly for 3 consecutive weeks). Mice were then treated with porcine bile extract (10mg/mouse in 200μL water, 3 times weekly for 4 weeks) by gavage. Mice were sacrificed 24 hours after the last treatment and histopathological analyses in the esophagus, forestomach, squamocolumnar junction within the glandular stomach, and other tissues were performed. In wild type mice, only 20% of mice treated with NMBA/bile extract developed small papillomas in the forestomach and squamocolumnar junction at the stomach. In BK5.erbB2 mice, 50% of mice treated with bile extract alone developed significant hyperplasia, 63% of mice treated with NMBA alone developed papillomas, and 100% of mice treated with NMBA/bile extract developed large SCCs in these areas. BK5.erbB2 mice treated with bile extract also showed significantly elevated levels of cell proliferation as determined by 5-bromo-2′-deoxyuridine incorporation in esophagus and forestomach compared to wild type mice. These results indicate that bile acids may play an important role in the development of stomach/esophageal tumors where erbB2 is overexpressed and/or activated. In this regard, bile acids may promote the development of these tumors. Furthermore, these studies demonstrate the utility of the BK5.erbB2 mouse as a novel model for human stomach and esophageal cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4176.

Bile tolerant Lactobacillus reuteri isolated from pig feces inhibits enteric bacterial pathogens and porcine rotavirus

Lactic acid producing bacterial strain Probio-16 was isolated from the swine excrements under anaerobic conditions and characterized by morphology and biochemical characteristics. The strain was further identified by 16S rRNA gene sequencing and phylogeneitc analysis. The antimicrobial activity of the strain was assayed by testing for growth inhibition of thirteen pathogenic microorganisms. The strain was tested for antiviral activity against porcine rotavirus in vitro in African green monkey epithelial cell line TF-104. Antibiotic susceptibility of the strain against 13 antibiotics was tested using disk diffusion method. Phenotypically and through 16S rRNA gene sequences, Probio-16 was identified and named as Lactobacillus reuteri Probio-16. This strain was resistant to pH 2.0, 5% porcine bile and exhibited antimicrobial activity against all the thirteen enteric bacterial pathogens tested. Probio-16 supernatant inhibited porcine rotavirus in vitro in TF-104 cell lines. Except for erythromycin and penicillin G at a concentration of 4 microg/ml, Probio-16 showed resistance to all other thirteen antibiotics tested. This study indicates L. reuteri Probio-16 as a novel strain with its tolerance to low pH and bile, antimicrobial activity, antibiotic resistance and antiviral activity against rotavirus, and an ideal probiotic candidate for animal and human application after the proper in vivo experiments.

Probiotic potential ofStaphylococcus hominisMBBL 2–9 as anti‐Staphylococcus aureusagent isolated from the vaginal microbiota of a healthy woman

To isolate and characterize an antagonist for use as probiotic agent in the biocontrol of Staphylococcus aureus. Bacteria that exhibited antimicrobial activity against Gram-positive bacteria including Staph. aureus were isolated from 12 healthy women, with Staphylococcus hominis MBBL 2-9 showing the strongest activity. The bacteriocin produced by Staph. hominis MBBL 2-9 was purified by 60% ammonium sulfate saturation, ultrafiltration, HLB cartridge and reverse-phase HPLC. The molecular weight was estimated as 2038.2 Da by MALDI-TOF mass spectrometry. The antagonist survived up to 2 h in artificial gastric juice (pH 2.5) and grew in the presence of 1% porcine bile extract. In addition, Staph. hominis MBBL 2-9 adhered effectively to HT-29 epithelial cell line. Staphylococcus hominis MBBL 2-9 exhibited desirable probiotic traits such as acid tolerance, bile resistance and adherence to epithelial cell line. The bacterium also produced a bacteriocin with unique molecular weight and high antimicrobial activity similar to traditional antibiotics. This study is the first report of a bacteriocin-producing Staph. hominis MBBL 2-9 that has potential for use as a probiotic agent against Staph. aureus.

Holmium: YAG Laser Safety Data On Bile Duct Epithelium in the Porcine Model

Background: Laser therapy has been increasing used for gastrointestinal and urological applications. Due to a very short wavelength, holmium: YAG can generate high-energy focused shock waves while reducing surrounding thermal injury. This characteristic has led to recent interest in using this technology for the fragmentation of large bile duct stones that have been refractory to conventional endoscopic therapy. Aim: The aim of this study was to establish safety data for holmium: YAG laser lithotripsy within the bile duct in the porcine model. Methods: 6 female 50 kg domestic pigs underwent necropsy and the bile ducts were explanted. The bile ducts were excised longitudinally exposing the epithelial surface and submerged under tension in liquid media. A Slimline holmium YAG laser probe (Lumenis, Sunnyvale, CA) was placed in perpendicular contact with the bile duct epithelium and activated at various energy density settings to evaluate perforation rates. The power density settings ranged from 8 to 15 watts (W) and were accomplished using various combinations of frequencies (Hz) and joules (J). The laser was activated until perforation of the bile duct occurred or for a maximum of 60 seconds. For each setting the probe was placed in similar locations on both the proximal and distal aspects of the duct. Both location and wattage were randomized to avoid physiologic differences that may affect outcomes. The median perforation times at each power setting were then compared using the Kruskal-Wallis One Analysis of Variance. Results: 104 data points were collected from 6 porcine bile ducts using 6 different power settings: 8 W (n=22); 9 W (n=22); 10 W (n=16); 12 W (n=20); 14 W (n=8); 15 W (n=16). The median perforation times were: 8 W (60 s); 9 W (24.5 s); 10 W (28 s); 12 W (24.5 s); 14 W (14 s); 15 W (5 s). Pairwise multiple comparison (Dunn's Method) revealed statistically significant differences (P<0.05) in the perforation times between the following settings: 8 W vs 15 W; 9 W vs 15 W; 10 W vs 15 W; 12 W vs 15 W. Conclusions: Holmium: YAG laser appears to be safe when used in short bursts (< 5 sec) at power settings ranging from 8 W to 12 W. Further studies are required to determine the ideal power settings balancing effective stone fragmentation and perforation risk.